Runx2 is a scaffolding protein that interacts with proteins representing many different functional classes, including chromatin remodeling factors, proteins coupled to cell growth control, differentiation of osteoblasts and production of bone matrix, as well as those proteins that transduce developmental signaling pathways for bone formation. We have established that Runx2 recruits to its subnuclear domains associated with the nuclear matrix, intracellular mediators of signaling pathways that are both positive and negative regulators of bone formation, including Smads in response to BMP/TGF(3, and YAP, a WW domain protein in response to Src signaling. We have defined specific point mutations in the Runx2 protein that can disrupt these critical interactions between Runx2 and Smad and Runx2 with WW domain proteins, which include a growing number of factors that influence Runx2 activity on target genes. These point mutations allow us to address the in vivo significance of these interactions in nuclear microenvironments in mouse knock-in models. Our discovery of miRNAs that affect osteoblast differentiation leads us to address how micro-RNAs (miRNA) that target Runx2 and Runx2 co-factors regulate bone formation through modification of the proteins that form Runx2 coregulatory complexes in the nucleus. Project 2 will now pursue how these multiple signaling pathways which converge on Runx2 are regulated during osteoblast differentiation for the control of bone formation. We will 1) characterize Runx2-Smad target genes and regulatory complexes required to complete the BMP2 osteogenic signal;2) characterize the biological mechanisms and signaling pathways influencing the organization of WW coregulatory proteins with Runx2 to control osteogenesis;and Aim 3) investigate how miRNA candidates that target Runx2 and coregulatory factors regulate osteogenesis. Clinical Relevance: There is a pressing need to develop anabolic therapies for treating bone loss in osteoporosis from the aging skeleton or induced secondary to a metabolic bone disorder. Our studies will define novel targets that produce new bone in response to BMPs, shift the stem cell differentiation towards the osteoblast lineage and identify miRNA regulators of bone formation. Each of these represents potential therapeutic applications to stimulate osteoblast differentiation and bone formation. For example, siRNA and miRNA technologies are being developed for in vivo application. Anabolic therapies that are safer than hormone treatments could be developed for stimulating bone formation.